Gene therapy is a technique which operates by delivering genetic material to endogenous cells within a functioning human or non-human animal body to add additional DNA-driven capacity to those cells, for example to introduce novel genes, to introduce additional copies of pre-existing genes, to impair the functioning of existing genes, or to repair existing but non-functioning genes. To some extent it is comparable to transfection of cells in a cell culture, but since the cells to be treated are part of the complex whole of the animal body, the overall functioning of which must be maintained, gene therapy faces particular problems in terms of the mechanism of delivery of the genetic material to the cells to be treated. To this end, carriers have been used to protect the genetic material from degradation and to facilitate its cellular uptake.
One line of approach has been to take advantage of the life cycle of viruses, which transfer their genetic material into host cells and co-opt the functions of the host cells (or their progeny) to replicate and release copies of the virus. Accordingly viral vectors have been used to transfer desired genetic material into host cells in the animal. Nonetheless, viral vectors have limited capacity to carry bulky DNA and moreover there are concerns over the safety of using viral vectors in gene therapy, especially of humans (see for example Danielsen et al., Biochimica et Biophysica Acta 1721: 44-54 (2005)). In 1999, for example, 18-year old Jesse Gelsinger, who was participating in a gene therapy trial for ornithine transcarboxylase deficiency, died from multiple organ failure thought to have been triggered by an immune response to the adenovirus vector that was being used.
Apart from direct injection into the cells to be treated and localised injection coupled with electroporation of the target cells, both of which are of limited utility, there are two primary categories of non-viral delivery systems for genetic material for gene therapy, namely lipoplexes and polyplexes. Lipoplexes are liposomes formed by mixing cationic lipids with anionic DNA and contain the DNA within a protective lipid shell (see for example Torchilin, Nature Reviews Drug Discovery 4: 145-160 (2005)). Polyplexes are nanometer-sized self assembled particles comprising a polycationic polymer and the anionic DNA—the polycationic polymer compacts the DNA to form nanoparticles in which the DNA is at least partially protected from enzymes, e.g. extracellular nucleases, which would otherwise degrade it. (See for example De Smet et al., Pharmaceutical Research 17: 113-126 (2000)).
Both lipoplexes and polyplexes are formed by electrostatic interactions and have a surface net positive electric charge. This net charge has the advantages of preventing the particles from clumping into aggregates and assisting in promoting cellular uptake, but has the disadvantage that the particles are vulnerable to undesired electrostatic interaction with endogenous anionic extracellular biomolecules (see Remaut et al., Materials Science and Engineering 58: 117-161 (2007)).
Indeed, in general, unless the genetic material can be injected directly into the target cells, which, as mentioned, is of limited feasibility in gene therapy, the genetic material faces a host of barriers between administration and cellular uptake. If administered alone, it is liable to destruction before uptake, for example by extracellular nucleases, while if it is administered as a complex that complex must overcome such barriers as blood, interstitium and mucosal layers before genetic material uptake occurs. While viruses naturally have systems for overcoming these barriers, as mentioned above there are serious problems associated with viral vectors. These barriers, or “extracellular matrices”, and their components may dramatically alter the surface properties of the DNA complexes, potentially inducing their aggregation or destruction, or causing their immobilization, or altering their ability to undergo cellular uptake upon arrival at the target cells. Such interactions, moreover, may activate the complement system and immune response, further reducing the chances of successful, side-effect free, delivery of the genetic material to the target cells.
As a result, surface coating of the complexes with camouflaging materials, such as PEG, which provoke little or no response has been proposed. This too, however, suffers from drawbacks. Thus, for example, lipoplexes prepared with PEGylated lipids tend to carry the DNA unprotected on their surfaces rather than inside, and so the liposomes must instead be PEGylated post-production. Additionally, PEGylation interferes with DNA release from lipoplexes and may cause generation of anti-PEG antibodies, which will limit therapy to a single round of treatment.
There thus remains a continuing need for an improved manner of delivery of genetic material for gene therapy.